Protein Identification

Suitable for simple protein sample enriched with a protein of interest. For example, a gel band cut from 1D SDS-PAGE or highly purifed protein sample

About Protein Identification

This service is for the identification of proteins from simple protein sample. There is one fractionation step by reverse-phase liquid chromatography. Normally, a hundred of proteins could be identified. For extensive protein identification, please refer to our proteomic profiling service which adapt two dimensional fractionation procedures.
Keeping sample simple could reduce the suppression effect from others irrelevant peptides to the peptides derived from the protein of interest during MS analysis. The presence of other interfering proteins as in complex protein sample leads to two issues:
  1. Reduce the homogeneity of each chromatographic fraction and thus increase the overlapping potential of the isotopic envelope of interfering peptides with that of the peptides derived from the protein of interest
  2. MS2 spectrum become much noisy and hence reduce the accuracy of peptide identification
Protein Digestion

Protein Digestion

Proteins are digested with proteolytic enzyme.

Peptide Separation

Peptide Separation

Digested peptides are fractionated by RPLC

Tandem MS Analysis

Tandem MS Analysis

Each fraction is on-line injected into MS

Data Analysis

Data Analysis

Peptide sequences are obtained from MS2 spectra

 

With the use of high resolution LC/MS/MS, the exact sequence of a protein can be revealed on MS2 spectrum, Figure 1 and 4. In typical experimental workflow, the protein of interest is first digested and downsized with proteolytic enzyme into small peptides which are more readily resolved by mass spectrometer. These peptides are then separated based on their hydrophobicity in a reverse phase liquid chromatography column. Each chromatographic fraction is analyzed by MS. In MS1, the peptides in a fraction are separated based on their mass-to-charge ratio to obtain MS1 spectrum. Given sufficient resolution of mass spectrometer and free of other interference, an elegance isotopic envelope of a peptide could be observed, Figure 2. If the peptide has low molecular mass, the most abundant peak would be the monoisotopic peak. In data-dependent acquisition mode, the most abundant peak/peaks would be selected for further fragmentation. If collision induced fragmentation method is chosen, the monoisotopic peptide would be fragmented into a series of y- or b- ions, Figure 3. These ions are then separated in MS2 based on their masses. By calculating the mass difference between adjacent y- or b- ions respectively, the peptide sequence could be obtained as amino acids have different exact masses (except isoleucine and leucine), Figure 4

 

 

Figure 1. Peptide sequence is revealed on MS2 spectrum.

Figure 2. Typical isotopic envelope of peptide.

Figure 3. CID produces a series of y- and b-ions.

Figure 4. Peptide sequence can be obtained by comparing the mass difference between adjacent y- or b-ions respectively with monoisotopic masses of amino acid

Service Package

In-solution/gel digestion

LC/MS/MS Analysis

Raw Data Export and Conversion

Protein Identification

Free Protein Quantification

Generation of Analysis Report

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Notes

  1. Sample submitted should contains at least 2ug of proteins. The sample could be submitted either as Coomassie Blue stained 1D or 2D gel band or as dry protein powder. Make sure the protein sample is sufficiently desalted before submission
  2. Sample is analyzed by Waters QTOF/ Thermo LTQ-Orbitrap/ AB Sciex 5500
  3. Proteins are identified by MASCOT® database search
  4. Intensity weighting, normalization, bias and isotopic overlap correction will be applied when appropriate
  5. Free protein quantification is applicable to sample that is labeled with stable isotopes (e.g. 15N metabolic labelling or SILAC)
  6. If more than one sample are submitted, free label-free quantification service is offered.

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